An integrated classification of tumor suppressor IKZF1 inactivation and oncogenic activation in Philadelphia chromosome‐like acute lymphoblastic leukemia

Abstract Philadelphia chromosome (Ph)‐like acute lymphoblastic leukemia (ALL) is recognized for its genetic and clinical diversity. In this study, we identified a novel high‐risk subset of Ph‐like ALL, characterized by the activation of oncogenic signaling and the inactivation of the tumor suppressor gene IKZF1, resulting in a dismal outcome. The association between cytogenetic aberrations and clinical features was assessed on a cohort of 191 patients with Ph‐like ALL. Our findings revealed that patients with inactivation of IKZF1 combined with activation of oncogenic signaling (CRLF2/EPOR/JAK2 rearrangements or p‐CRKL/p‐STAT5 high expression) had the worst outcome (3‐year overall survival [OS] of 28.8% vs. 80.1% for others, p < 0.001; 2‐year event‐free survival [EFS] of 6.5% vs. 57.0% for others, p < 0.001). Multivariable analysis demonstrated that this high‐risk feature was an independent inferior prognostic factor (adjusted hazard ratio for OS = 4.55, 95% confidence interval [CI]: 2.35–8.81, p < 0.001; adjusted hazard ratio for EFS = 3.27, 95% CI: 1.99–5.39, p < 0.001). Allogeneic hematopoietic stem cell transplantation was associated with improved prognoses in patients within the high‐risk subgroup. In conclusion, this study identified a clinically distinct entity that possesses effective prognostic features and provides potential guidance for refining risk stratification in Ph‐like ALL.


INTRODUCTION
][3] Despite improvements in outcomes with tyrosine-kinase inhibitors (TKIs) for patients with ABL-class Ph-like ALL, 4 the overall prognosis for Ph-like ALL remains inferior, with a 5-year event-free survival (EFS) rate of 22.5% and an overall survival (OS) rate of 23.8%, compared to 50%-60% OS observed in other subsets of Ph-negative (Ph − ) ALL. 3 Therefore, it is crucial to identify the subsets with the worst outcomes within the highly diverse landscape of Ph-like ALL to develop risk-directed strategies.The oncogenic pathways involved in Ph-like ALL predominantly comprise the ABL-class, CRLF2, and JAK-STAT signaling, which are associated with pro-proliferative or anti-apoptotic properties and are part of the current risk classification. 5,6Moreover, tumor suppressor gene (TSG) IKZF1 deletions have emerged as the most important cytogenetic aberrations in B-cell ALL (B-ALL).They are frequently observed in approximately 50%-60% of Ph-like ALL cases.][9][10][11][12] In addition, IKZF1 plus (defined as IKZF1 deletions that co-occur with deletions in CDKN2A, CDKN2B, PAX5, or PAR1 in the absence of an ERG deletion) is associated with worse outcomes in pediatric patients with B-ALL 8 and has been incorporated into the 2023 National Comprehensive Cancer Network (NCCN) guidelines.However, the prognostic implications of combining IKZF1 aberrations and oncogenic activation in Ph-like ALL have been marginally addressed.This study characterizes a novel high-risk (HR) subgroup of Ph-like ALL, which comprises a combination of IKZF1 dysregulation and activated oncogenes related to Ph-like ALL, which stratifies patients with Ph-like ALL into higher-and lower-risk categories.The new stratification may lead to innovative and effective targeted treatments for Ph-like ALL.

Study design
This multicenter, retrospective cohort study was designed to explore the clinical features of patients with Ph-like ALL in the South China Hematology Consortium (SCHC).Patients with Ph-like ALL were enrolled in five SCHC centers between September 2016 and December 2021.A cohort of patients with Ph − ALL diagnosed at Nanfang Hospital (NFH) between January 2016 and December 2021 was introduced for comparison of survival.Informed consent was obtained from all the patients.This study was reviewed and approved by the Ethics Board of Nanfang Hospital, in accordance with the Declaration of Helsinki.The final follow-up was conducted in September 2022.The diagnosis of Ph-like ALL was generally based on the Chinese guidelines, NCCN guidelines, and World Health Organization (WHO) classification. 5,6,13The diagnosis criteria included ABL-class fusions, CRLF2 rearrangement or high expression, JAK-STAT pathway alterations, Ras pathway mutations, and phosphorylated (p)-CRKL or p-STAT5 high expression.Patients received chemotherapy or targeted drugs (TKIs and ruxolitinib) along with chemotherapy as frontline treatment.The chemotherapy regimens included Precision-Classification-Directed-Target-Total-Therapy (PDT)-ALL-2016 11,14,15 (for NFH) and the Chinese guideline for the treatment of adult ALL 5 (for the other four SCHC centers).Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was recommended for patients who achieved complete remission (CR) if a donor was available.Salvage therapies, including intensive chemotherapy, targeted drugs, or immunotherapies (including blinatumomab and CAR-T) were administrated to patients with minimal residual disease (MRD)-positivity after induction and relapsed/refractory (R/R) disease.

Statistical analysis
Categorical data was summarized by the proportion of patients.Quantitative data was described using median for central tendency and range or interquartile range (IQR) for distribution range.The χ 2 and Fisher's exact test were used to compare categorical variables.The t-test and the Mann-Whitney U test were used to compare continuous variables.The Kaplan-Meier method was used to assess survival data using the log-rank test.OS was estimated from the date of diagnosis to the date of last follow-up or death from any cause.EFS was estimated from the date of diagnosis to the date of the last follow-up or the first event (including nonresponse after induction, relapse, and death from any cause).To compare the prognosis of patients receiving allo-HSCT or not, leukemia-free survival (LFS) was estimated from the date of 6 months after diagnosis to the date of the last follow-up, relapse, or death from any cause.The Cox proportional hazards regression model analyses including univariable and multivariable analyses were used to identify potential prognostic factors associated with OS and EFS.Statistical significance was defined as a two-sided p-value of <0.05.All statistical analyses were performed using R software (version 4.2.0, https://cran.r-project.org/).

Clinical characteristics
A total of 191 adolescent and adult patients with Ph-like ALL were included in the analysis (Figure 1), and their main clinical characteristics are summarized in Supporting Information S1: Table S1.The Ph-like ALL cohort comprised 74 females and 117 males, ranging from 14 to 71 years, with a median age of 26 years (IQR 18-36)  rearrangement and CRLF2 high expression.Patients with EPOR/JAK2 rearrangement had higher WBC counts at diagnosis than those in other subgroups.A total of 68 patients (35.6%) received anti-CD19 and/or CD22 CAR-T therapy and 115 patients (60.2%) received allo-HSCT.The other Ph − ALL cohort included 155 patients with a median age of 28 years at diagnosis (range, 14-69 years).Among them, 97 (62.6%) underwent allo-HSCT (Supporting Information S1: Table S1).
Activation of CRLF2/EPOR/JAK2/p-CRKL/p-STAT5 plus inactivation of IKZF1 exhibited the worst outcome To identify the subgroup with the worst outcomes, we evaluated the combined effects of oncogenes activation and IKZF1 inactivation in patients with Ph-like ALL.A total of 166 patients with available IKZF1 data were included in the subsequent analyses.To explore potential combinations associated with poor prognosis, we combined different molecular subtypes with IKZF1 alterations to calculate the risk ratio for event occurrence (Supporting Information S1: Table S5).Considering that high p-CRKL and p-STAT5 expression may occur simultaneously with other molecular abnormalities, the cases included in the analysis of p-CRKL/p-STAT5 combined with IKZF1 were those exhibiting p-CRKL or p-STAT5 high expression, regardless of any co-occurring molecular abnormalities.A statistically significant combination indicated a potentially HR subgroup in Ph-like ALL, and all these combinations were merged into a new combination, namely CRLF2/EPOR/JAK2/p-CRKL/p-STAT5 F I G U E 2 Genomic landscape of Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL).Data are presented for 191 patients with Ph-like ALL, categorized based on specific genetic abnormalities.The categories include patients with ABL-class fusions (ABL1, ABL2, CSF1R, and PDGFRB), CRLF2 positive (rearrangement or high-expression), EPOR or JAK2 rearrangements, other JAK-STAT pathway mutations (IL7R, FLT3, SH2B3, JAK1, JAK3, and TYK2), Ras pathway mutations only (KRAS, NRAS, PTPN11, NF1, BRAF, and CBL), and p-CRKL/pSTAT5 high-expression only.Alterations in transcription factors (IKZF1, PAX5, EBF1, and ETV6) are also shown.For specific details regarding fusion partner genes and mutations, please refer to Supporting Information S1: Table S3 and Supporting Information S1: Figure S2.p-CRKL, phosphorylated CRKL; p-STAT5, phosphorylated STAT5.
combined with IKZF1 alterations.The new combination was classified as a HR subgroup, whereas the remaining subgroup was classified as a standard-risk (SR) subgroup.Patients within the HR subgroup had worse prognoses than those within the standard-risk subgroup (3 y-OS 28.8% and 80.1%, respectively, and 2 y-EFS 6.5% and 57.0%, respectively; Figure 3E,F).The median time from the first CR to relapse was 5 months (IQR: 1-11, HR) and 13 months (IQR: 4-25, SR), respectively.There were no significant differences in the proportions of treatments that patients received between HR and SR subgroups (Supporting Information S1: Table S6).Stratified subgroup analysis also revealed a similar trend of worse outcomes in HR than that in SR (Figure 4 and Supporting Information S1: Figure S3).Sensitivity analyses showed that dividing the cohort into NFH and the other four SCHC centers did not markedly affect the results (Supporting Information S1: Figure S4).Because the p-CRKL/p-STAT5 subgroup was not strictly Ph-like ALL, it was excluded and re-analyzed.The combination of CRLF2/EPOR/JAK2 and IKZF1 still exhibited an unfavorable prognosis when excluding p-CRKL/p-STAT5 (Supporting Information S1: Figure S5).Within the subgroup of CRLF2/EPOR/JAK2, IKZF1 alterations were associated unfavorable prognosis (Supporting Information S1: Figure S5).Cox model analysis was performed to assess the prognostic factor for OS and EFS.There were statistically significant differences in outcomes between the HR and the SR subgroup (adjusted hazard ratio for OS = 4.55, 95% confidence interval [CI]: 2.35-8.81,p < 0.001, Table 1; adjusted hazard ratio for EFS = 3.27, 95% CI: 1.99-5.39,p < 0.001, Supporting Information S1: Table S2).In addition, within the HR subgroup exhibited adverse prognoses compared to the Ph − ALL cohort, whereas SR and other Ph − ALL showed comparable prognoses (Supporting Information S1: Figure S6).Finally, because the measurable residual disease (MRD, examined by flow cytometry with a sensitivity of 10 −4 ) status has significant prognostic significance in the Cox model analysis, the prognostic effect of the HR feature in the context of treatment response was assessed.In patients with MRD-positive after induction, outcomes were affected by the presence of HR feature, whereas outcomes of patients with MRDnegative after induction were not significantly affected by HR feature (Supporting Information S1: Figure S7).

Immunotherapies and allo-HSCT in patients within the HR subgroup of Ph-like ALL
Then we focused on evaluating the effectiveness of immunotherapies and allo-HSCT in improving the prognosis of patients within the HR subgroup.Among the 43 patients exhibiting HR feature, four (9.3%) received blinatumomab.Of these, two underwent treatment due to induction failure, one with CR received treatment post-induction to improve prognosis, while one was treated following relapse after transplantation.Both patients who received blinatumomab due to induction failure died of disease progression, whereas the other two patients remained alive.Additionally, 16 (37.2%) of the 43 HR patients underwent CAR-T therapy, with a median time from diagnosis to CAR-T administration recorded at 5 months (IQR: 3-15).Notably, all 16 patients achieved CR post-CAR-T administration, but eight experienced relapses, with a median time to relapse of 4 months (range 2-16).Furthermore, 21 (48.8%) of the 43 HR patients underwent allo-HSCT, with a median time from diagnosis to allo-HSCT of 6 months (IQR: 5-7), comprising 20 at CR1 and one at CR2. Nine of the 21 (42.9%)patients received CAR-T prior to transplantation.A landmark analysis, which included only patients alive and in remission at 6 months after diagnosis, was conducted to evaluate post-allo-HSCT outcomes.A total of 26 patients were included in this analysis.Undergoing allo-HSCT was associated with better LFS and OS (Figure 5).

DISCUSSION
Ph-like ALL is characterized and classified based on the activation of multiple oncogenes, such as ABL1, CRLF2, and EPOR rearrangements, leading to a heterogeneous disease phenotype.Meanwhile, Ph-like ALL shares a high frequency of TSG IKZF1 deletions with the Phpositive subset.IKZF1 aberration is considered as a prognostic factor in B-ALL, particularly in Ph-positive and Ph-like ALL, leading to adverse outcomes.In Ph-positive ALL, IKZF1 deletions contribute to leukemogenesis and TKIs resistance in conjunction with BCR::ABL1 fusion. 18,19In pediatric B-ALL, the AIEOP-BFM group defined F I G U R E 4 Subgroup analysis of overall survival (OS) in 166 patients.A total of 166 patients were divided into two groups, HR and SR, for subgroup analysis.Data are n (%).IKZF1 plus CD20 was defined as IKZF1 deletions with CD20-positive (≥20%).Targeted drugs included TKIs (imatinib, dasatinib, and orelabrutinib) and JAK2 inhibitor (ruxolitinib).HR, high-risk; SR, standard-risk.
IKZF1plus as IKZF1 deletions combined with CDKN2A, CDKN2B, PAX5, or PAR1 deletions without an ERG deletion, which confers the worst outcomes. 8Deletion of 22q11.22 is also common in pediatric Ph-like ALL, and the combination of 22q11.22 and IKZF1 deletions can refine the risk stratification. 20In adult B-ALL, our previous study demonstrated that IKZF1 deletions plus CD20 positivity correlated with a very poor prognostic profile. 11These findings characterize the subgroups with poor prognoses in B-ALL and refine the IKZF1-based prognostic stratification.In the current study, we revealed a synergistic effect of CRLF2/EPOR/JAK2/p-CRKL/p-STAT5 and IKZF1 in Ph-like ALL, which resulted in poor outcomes and shaped the Ph-like classification into HR and SR subgroups.
The current classification of Ph-like ALL is inadequate for predicting patient outcomes.The re-classification of HR and SR subgroups enabled the identification of patients with poor prognoses, which could be valuable for risk classification and seeking more effective targeted treatments.Although gene expression profiling analysis is the standard method for diagnosing Ph-like ALL, 1,21 it remains crucial to develop more effective diagnostic approaches.We employed a comprehensive screening strategy involving flow cytometry, fluorescence in situ hybridization, reverse-transcription polymerase chain reaction, targeted next-generation sequencing, RNA sequencing, and whole-exome sequencing.Furthermore, the challenge of treatment lies in bridging the between the identification of novel subsets and the lagging development of targeted strategies.Optimal therapeutic approaches for Ph-like ALL have not yet been clearly characterized.ABL-class Ph-like ALL is sensitive to TKIs, 2 and our results showed that this subgroup had a relatively favorable prognosis in Ph-like ALL.JAK2 inhibitor ruxolitinib showed promising results but with high doses.Other targeted drugs, such as Ras and MEK inhibitors in the Ras class, remain far from bench to bedside. 22In addition, immunotherapy bi-specific CD3/CD19 antibodies blinatumomab has shown promising results in R/ R and CRLF2-rearranged Ph-like ALL. 23,24However, resistance remains a challenge, especially in patients with IKZF1 deletions and IKZF1 plus. 9t is worth mentioning that combining Hyper-CVAD and blinatumomab in the frontline setting has led to improved outcomes, with a 3-year OS rate of 76% in patients with high-risk features (including CRLF2 and TP53). 25As for CAR-T therapy, a significant portion of patients (42.9%) received CAR-T prior to transplantation.Therefore, the role of CAR-T therapy in HR subgroup cannot be dismissed completely, warranting further investigation.Finally, allo-HSCT remains crucial for achieving favorable survival outcomes for Ph-like ALL. 26,27In this study, allo-HSCT was associated with improved outcomes in patients within the HR subgroup of Ph-like ALL.However, given that the small numbers of  patients (n = 26) included in the analysis, these findings should be interpreted with caution.Although transplantation may improve the prognosis of patients with HR Ph-like ALL to some extent, their prognosis remains worse compared to the SR subgroup.This disparity may be attributed to the lack of targeted therapies for oncogenes like CRLF2, EPOR, and JAK2, particularly for inactivated TSG IKZF1.IKZF1 alterations will lead to acquisition of stem cell-like features, metabolic reprogramming, and resistance to drugs and immunotherapies, 7,9,10,16,17,[28][29][30] underscoring the importance of targeting IKZF1 deletions in Ph-like ALL.In fact, there is still a lack of knowledge regarding how to target IKZF1 deletion ALL.We previously reported that tucidinostat targets IKZF1 deletions ALL by restoring IKZF1 expression in vitro and in vivo. 10In the current study, we identified a novel subgroup with poor outcomes characterized by IKZF1 alterations combined with CRLF2/EPOR/JAK2 rearrangements or p-CRKL/p-STAT5 high expression.This suggests the necessity of a dual-targeting approach for IKZF1 and oncogenes like CRLF2, EPOR, and JAK2 in the HR subset, and further translational research on underlying mechanisms is necessary.
This study has several limitations.First, the definition of the HR subgroup included p-CRKL/p-STAT5 only measured in NFH, which might limit the generalizability of the findings.Second, previous studies suggested that patients with CRLF2 overexpression have worse outcomes, 31 whereas our study found noninferior outcomes in patients with CRLF2 high expression.This inconsistency may be attributed to variations in the criteria for defining CRLF2 high expression, differences in the frequency of IKZF1 alterations or JAK2 mutations, and potential racial and ethnic disparities.Third, IKZF1 status data were not available for 25 of the 191 patients.These cases were excluded from the analysis of HR and SR classification, which could have introduced a potential bias.Moreover, due to a lack of systematic assessment of abnormalities of IKZF1 plus, 8 this study did not include IKZF1 plus, which may be relevant to the adverse outcomes of patients within the HR subgroup.Finally, as an observational study, there is the risk of unmeasured confounding factors that may counteract the apparent contribution of the HR feature as an independent risk factor for Ph-like ALL.
In conclusion, our study identified a clinically distinct HR subgroup that serves as a valuable prognostic feature of Ph-like ALL and may provide guidance for refining Ph-like ALL risk stratification for cutting-edge therapeutic approaches.
at diagnosis.The most common subtype was CRLF2, which was observed in 52 of 191 patients (27.2%) and included CRLF2 F I G U R E 1 Study profile.HR, high-risk; NFH, Nanfang Hospital; SCHC, South China Hematology Consortium; SR, standard-risk.HemaSphere | 3 of 9

3
Overall survival (OS) and event-free survival (EFS) of different subgroups in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL).(A) OS and (B) EFS of 191 patients with different subtypes of Ph-like ALL; (C) OS and (D) EFS of 166 patients stratified by IKZF1 status; (E) OS and (F) EFS of 166 patients divided into HR and SR subgroups.HR, high-risk; SR, standard-risk.HemaSphere | 5 of 9